A number of patents exist for various forms of suspended microarray assays, including patents for substrate microparticles of various kinds, how their sets are differentiated, processing the array data and what is termed “multiplexing” of assays. A list of these references is submitted in an Information Disclosure Statement accompanying this application. A number of these patents are held by the Luminex® corporation, a company that produces specialized flow cytometers for use with microsphere based suspended microarray assays.
Of those patents that cover multiplexing, none discuss the intentional use of more than one particle set targeted at the same analyte within a sample. None discuss how one might make use of a multiplicity of sensitivities to the same analyte by a set of suspended microarray assays in order to better determine analyte concentration. None disclose using a multiplicity of assays that should have the same sensitivity to analyte to improve precision. Nor do any of the listed patents discuss how both techniques (multiple identically sensitive assays and multiple differentially sensitive assays for the same analyte) can be combined together to achieve an even higher level of precision and repeatability. None of the patents discuss how such intraplexed assays can be used to eliminate instrumentation variances and make accurate estimates of true concentration of analytes without necessarily requiring calibration standards be run with every multi-well assay that is performed.
To define intraplexing clearly, special terminology has been created because without it, experts became confused as to what exactly was being referred to. For this reason, a single particle (which is, for Luminex®, a microsphere) is called an SMP (suspended microarray particle.) A set of microspheres that are all labeled with the same classifier is called an SMPCS (suspended microarray particle category set). (Hence, SMPCSs is the plural form of SMPCS.) These two differentiations are all that is necessary for understanding suspended microarray systems. An SMPCS corresponds to what Luminex® commonly calls “a microbead region”, a “microbead set” or more colloquially, “a microbead” and is usually interchangeable with “bead number”, since Luminex® classifies their beads to users by numbers from 001 to 100. A method of classification of such sets will sometimes be referred to herein as a “classifier.”
Intraplexing also introduces a superset, which is the “suspended microarray particle category set—identical group,” or SMPCS-IDG. (Likewise SMPCS-IDGs is the plural form.) In an SMPCS-IDG, a set of differently numbered microparticle sets are all coated with the same reagent(s) so as to make them identical in sensitivity to the analyte being assayed. Bearing this introduction in mind, these terms are discussed in more detail below.
A suspended microarray system uses a population of suspended microarray particles (SMPs), all of which have one assay on their surface. These SMPs are run through a flow cytometer after running an assay protocol. The flow cytometer has a flow cell which differentiates individual SMP events as they go by. Thus, the result is a statistical sampling of the population of the SMPCS made up of individual readings. These individual event readings are not identical, but are distributed in an approximate normal curve. Conventionally, most of these SMP-based assays use fluorescent reporter molecules to provide a signal, but there are other methods. If multiple SMPCSs are present in a well, then more than one analyte can be assayed simultaneously in the same assay plate well. This is termed multiplexing of assays, and it is a primary selling point for current suspended microarray systems.
A multiplexed assay comprises different suspended SMPCSs—where each SMPCS has an assay for a different analyte, and the SMPCSs are mixed together in one test tube or assay plate well. Conventional assay plates currently contain up to 96 wells in one plate, wherein each well contains fluid to be assayed, as shown in prior art FIG. 5. In use, each of the 96 wells in the plate is loaded with some analyte in a fluid medium. Into each well are injected suspended microarray particles with assays on them, and then an assay protocol is performed. Finally, the 96 well plate is inserted into a robotic sampler that feeds a reading instrument. Typically, the reader is a flow cytometer. The robotic sampler inserts a hollow probe, column by row, one well at a time, sucking up a sample of fluid mixed with SMP's, moving the sample acquisition probe from one well to the next.
Another assay method is an environmental assay method sometimes known as “smart dust” which consists of a tiny electronic chip, (that can be microscopic) that has an assay built into it, generally on its surface. This assay device usually receives energy from the environment in the form of sunlight, or from a reader device in the form of microwaves. The device acts as a transponder, sending the result of the assay to the reader device. Some of these devices work in fluid, while others broadcast the particles (“smart dust”) over a region which may be outdoors. These broadcast assays indicate to the reader what is present in the environment. There are many identical copies of each assay particle in a broadcast system, and the signals returned can be analogous to signals returned by microspheres in a flow cytometry system, having many separate readings for the analyte that are combined to produce one value. In an environmentally broadcast assay system of this kind the equivalent of the test tube or well in a plate is the responding region containing particles that fall within an area the reader can detect. As the reader is moved, this area changes as particles are added on the leading edge of movement, and lost on the trailing edge. Totalizing of readings for a region can occur by making arbitrary distinctions between one such region and the next. In this type of system, as with fluid suspensions, a set of SMPCS assays could be created where each assay reacted to the same analyte. The results could be collated and processed in an analogous manner to that of intraplex assays taking place within a fluid medium.
One typical flow cytometry system for reading assays is the Luminex® system. To compensate for highly fluctuating readings, it is necessary that multiple wells be replicated for identical samples so that any variation may be seen. Typically this is done with two wells containing identical analyte samples, but this is of little statistical significance, because the “student t test” (hereinafter either “t test” or “t values”) value for a sample of just 2 has a multiplier of more than 60 on the standard deviation (at 99% confidence level) to get margin of error as shown in FIG. 4B. A t value is the statistical multiplier used on standard deviation to decide how wide the margin of error should be for sample sizes of 30 or less. (Use of t test rather than statistics appropriate for samples of more than 30 is not a limitation of this application, rather, for practical reasons, most of the time numbers will be in the t test range.) In part because statistically the accuracy does not improve much when two wells are used, and three wells costs as much as ELISA, most usually, for efficiency and simplicity, only one well samples are performed. One method routinely used for improving the precision and statistical significance employs a number of replicated sample wells coupled with 3 or more pairs of wells as standards, the standards having an increasing level of dilution so as to define a ratio that will correspond to analyte concentration. See FIG. 5. However, using replicates cuts into the cost effectiveness of suspended microarray assays, and can even make them more expensive than ELISA assays. Based on recent findings regarding the above-indicated lack of precision, using even more SMPs has been proposed by Luminex® to further increase precision. See Jacobson J W, Oliver K G, Weiss C, Kettman J. Analysis of Individual Data from Bead-Based Assays (“Bead Arrays”). Cytometry Part A 2006;69A:384-390. However, as described below, experiments have shown this method to have significant problems.
In multiplexing, the primary problem with using radically more SMPs for each SMPCS is that when assays are multiplexed there will be a statistical distribution of SMP counts obtained for each SMPCS in the multiplex. Theory predicts this effect, and experiment confirmed it. Thus it was found that to get a fairly reliable number of SMPs for each SMPCS when they are multiplexed an increasing number of SMPs for each SMPCS needs to be put into each sample well. This number of SMPs per SMPCS rises nonlinearly with the number of SMPs one attempts to collect for each SMPCS. In order to explain the problem better, an illustrative metaphor will be used of a swimming pool filled with M&Ms of different colors. In this example, the swimming pool is analogous to a single-sample well, and one M&M is analogous to an SMP. The populations of M&Ms that are of the same color are analogous to an SMPCS.
For this thought experiment, a swimming pool filled with M&Ms of 100 different colors is considered. An equal number of M&Ms of each of 100 colors is present and it is assumed the M&Ms are randomly mixed having no artifacts such as differential density of one color M&M leading to concentration at the bottom or top. A barrel of M&Ms is randomly scooped from the random assortment in the pool, and from the barrel, a large coffee can full of randomly scooped M&Ms is taken. Finally, all M&Ms in the coffee can are thrown high into the air, and those that land within an arbitrary 6-foot diameter circle are categorized. This 6 foot diameter circle corresponds to what is read by a flow cytometer that is able to categorize by color.
What will be seen from the above thought experiment is that there will not be an equal number of each color of M&Ms in the 6-foot circle. If one collects the counts of each color, and then categorizes them into a histogram with 5 to 20 different bins, plotting number of M&Ms in the histogram bin on the X axis against number of M&Ms per color for each bin, what would be seen is an approximation of a multinomial distribution of counts. Assuming the coffee can holds approximately 50,000 M&MS, FIG. 4A depicts a graph of the expected multinomial distribution. It is assumed in this example that 500 M&Ms of each color are desired.
What this multinomial distribution graph shows is that for about 30% of the colors, the number of M&Ms for each color will be near 500. But for some the counts will be much higher, and for some much lower. See FIG. 4A. In order to make reasonably sure that for 95% of the colors, 500 M&Ms are obtained, it is necessary to use a much larger coffee can. This graph also shows that the coffee can holding 50,000 M&Ms will be correct for a large-scale multiplex that has a minimum of 100 M&Ms of each color. What is not shown in this graph is that if the trials are repeated many times, once in a while outliers on both ends may appear, with even lower counts, or else very high counts.
In the case of the Luminex® flow cytometer, we know that the above “trial” will be repeated many thousands of times in the lifetime of an instrument. Understanding the statistical problem encountered in the swimming pool thought experiment makes the problem of SMPCSs in a multiplex assay easier to understand.
One Luminex® instrument, the new FlexMAP 3D™ can differentiate up to 500 different microsphere classifiers. If one were to create a multinomial simulation graph for that many microsphere classifiers, the outliers would be farther to the low and high regions of the graph. The intent of Luminex® in raising the number of classifiers is to allow the development of very highly multiplexed assays. However, if this capability is used, then it will show this distribution of counts problem even more than the current system that relies on 30 to 100 SMP reading events per SMPCS. It will require huge numbers of SMP's per SMPCS to work reliably.
There is thus a need to improve the precision of such assays without replicating samples, and to compensate for the multiple stochastic and non-stochastic sources of errors.
It is therefore a primary object of the invention to improve the precision with which each analyte can be read in any type of suspended array assay system by using a plurality of SMPCS readings for each assay.
It is a further object of the invention to provide a method to compensate for the multiple stochastic and non-stochastic sources of errors that can occur in this type of assay system.
It is a still further object of the invention to make possible statistically significant results from assays applied to single well samples. Since there is no statistical significance to a single result, (see FIG. 4B) and this method has more than one result, it represents an improvement over all assays in which a single result is used.
It is a still further object of the invention to make possible processed readings that have high correlation between instruments, even if the instruments have significantly varying responses to identical stimulus. Experiments have shown that instruments can vary significantly when reading exactly the same SMPCSs.
Other objects and advantages of the present invention will become obvious to the reader and it is intended that these objects and advantages are within the scope of the present invention.